The Wnt signaling pathway, mediated through active beta-catenin, is responsible for initiating the majority of cases of human colorectal cancer (CRC), and we have previously shown that hyper-activation of this pathway by histone deacetylase inhibitors (HDACis), such as butyrate, can induce the death of CRC cells. An important cellular switch that mediates the effects of Wnt signaling activation is variation in the association between beta-catenin and the transcriptional coactivators CREB binding protein (CBP) and p300. Association of CBP with beta-catenin is thought to activate a set of genes linked to cell proliferation, while the p300-mediated Wnt genetic program is believed to promote cell differentiation. Small molecule agents have been discovered that modulate CBP/p300 Wnt transcriptional programs by altering the association of CBP and p300 to beta-catenin. ICG-001 and ICG-427 inhibit CBP and p300 mediated Wnt activity, respectively, while IQ-1 prevents the shift from CBP-mediated to a p300-mediated Wnt activity. Aim 1 of this proposal is designed to determine the role of CBP and p300 mediated Wnt signaling in the response of CRC cells to HDACis. Cells will be cotreated with HDACis and ICG-001, ICG-427, or IQ-1 and the levels of Wnt activity, apoptosis, proliferation, differentiation, and CBP- or p300-beta-catenin binding measured. Aim 2 of this proposal is to determine the role of CBP/p300-mediated Wnt activity in the maintenance of high- and low-Wnt fractions in single CRC cell populations, which may mirror similar heterogeneity observed in human tumors and which may be of clinical significance. Aim 3 will compare the effects of CBP and p300 mediated Wnt activity on CRC initiation and progression, utilizing CRC cell line model systems of initiation and progression: the normal colon cell lines CCD-841CoN, the adenoma line LT97, the primary colon carcinoma cell line SW480, and the lymph node metastasis cell line SW620. Cells will be treated with HDACis and the small molecule agents, and assayed as described above. We will also attempt to use changes in CBP and p300 mediated Wnt signaling to shift colonic cells between cell type, modifying CBP and p300 mediated gene expression in the LT97 adenoma line to shift the adenoma phenotype to more characteristic of the CCD- 841CoN normal cells, or the SW480 carcinoma cells. We will utilize microarray analyses to determine the patterns of gene expression responsible for these CBP or p300 mediated changes in colonic neoplastic phenotype. The findings generated from this study will lead to future, more in-depth projects to further dissect the action of CBP/p300 Wnt-mediated transcriptional programs in colonic neoplasia, with an emphasis on methods to modulate these genetic programs for chemopreventive effect. PUBLIC HEALTH RELEVANCE: The Wnt signaling pathway is responsible for initiating the majority of cases of human colorectal cancer, and we have previously shown that hyper-activation of this pathway by histone deacetylase inhibitors (HDACis) can induce the death of colorectal cancer cells. Wnt signaling mediated by the proteins CBP or p300 differentially influence decisions of cancer cell growth, differentiation, or death, and small molecule agents exist that can specifically block CBP and p300 mediated Wnt signaling. Through the use of these small molecule inhibitors, we will determine how CBP and p300 influence colonic tumorigenesis;the findings of this study can be utilized to devise methodologies, targeting CBP and p300 mediated Wnt activity, to prevent the development and progression of human colorectal cancer, decreasing the morbidity and mortality associated with this disease.